High-throughput multiplexed assay for chemicals affecting neuron differentiation Neurodevelopmental disorders (NDDs) affect 400,000 to 600,000 of the 4 million children born in the US each year. The incidence rates for NDDs have been rising. Exposures to toxic chemicals may have contributed to NDDs. Due to neuronal development and maturation processes, fetus, newborns, and young children are especially vulnerable to neurotoxins at concentration levels that would not impact adult human health. In addition, children with genetic susceptibilities to NDDs may have higher risk than non-susceptible children. For neurodevelopmental toxicity, EPA and National Toxicity Program at the National Institute of Environmental Health Science rely on animal studies, which are inadequate for subtle neuronal changes, not human, lengthy in time, prohibitively costly with low-throughput. To address these limitations and to enable high-throughput assessment of chemicals' potential impact on human neuronal development, we propose an innovative gene expression profiling assay based on differentiating human induced pluripotent stem cells (iPSCs). iPSCs can be generated from people of various ethnicity and of diverse genetic background, which include those who are predisposed to neurodevelopmental and neurodegenerative diseases. JuvoBio Pharmaceuticals' scientific team consists of world-leading experts in using iPSCs-derived neurons to study brain development and disease mechanisms. We can reproducibly differentiate iPSCs into high-purity neuronal subtypes that are effected by ASD and other NDDs: FGNs (>90% purity), GABAergic neuron (>80%), and hippocampal neurons (>60%), in large-enough quantities to support high-throughput screen. By quantitatively assessing the expression level of key markers, we can identify the chemicals' impact on neuron differentiation. We will ascertain assay conditions that would allow the determination of increased susceptibility of genetically predisposed individual to chemical toxins. Our aims: Aim 1. Design probes and optimize compound treatment conditions. Using six known toxins as reference, we will optimize concentration titration, timing and length of treatment. Aim 2. Define the readout of the assay to predict impact on neuron differentiation. The impact of the neurotoxins on quantitative expression of up to 50 differentiation markers for both normal and autism iPSC lines will be analyzed for a smaller set of potentially predictive markers. Aim 3. Validate the readout using three alternative assays. Readout will be validated using Q-PCR, antibody staining, and mini-brain organoid assays.